Inhibition of inflammatory cytokine production by cholinergic agonists and vagus nerve stimulation

ABSTRACT

A method of inhibiting the release of a proinflammatory cytokine in a cell is disclosed. The method comprises treating the cell with a cholinergic agonist. The method is useful in patients at risk for, or suffering from, a condition mediated by an inflammatory cytokine cascade, for example endotoxic shock. The cholinergic agonist treatment can be effected by stimulation of an efferent vagus nerve fiber, or the entire vagus nerve.

RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 10/446,625,filed May 28, 2003 now U.S. Pat. No. 6,838,471, which is a continuationof U.S. Ser. No. 09/855,446, filed on May 15, 2001, now U.S. Pat. No.6,610,713, which claims priority to Provisional Application No.60/206,364, filed May 23, 2000. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to methods of reducinginflammation. More specifically, the invention relates to methods forreducing inflammation caused by proinflammatory cytokines or aninflammatory cytokine cascade.

2. Description of the Related Art

Vertebrates achieve internal homeostasis during infection or injury bybalancing the activities of proinflammatory and anti-inflammatorypathways. However, in many disease conditions, this internal homeostasisbecomes out of balance. For example, endotoxin (lipopolysaccharide, LPS)produced by all Gram-negative bacteria activates macrophages to releasecytokines that are potentially lethal (44; 10; 47; 31).

Inflammation and other deleterious conditions (such as septic shockcaused by endotoxin exposure) are often induced by proinflammatorycytokines, such as tumor necrosis factor (TNF; also known as TNF.alpha.or cachectin), interleukin (IL)-1.alpha., IL-1.beta., IL-6, IL-8, L-18,interferony, platelet-activating factor (PAF), macrophage migrationinhibitory factor (MIF), and other compounds (42). Certain othercompounds, for example high mobility group protein 1 (HMG-1), areinduced during various conditions such as sepsis and can also serve asproinflammatory cytokines (57). These proinflammatory cytokines areproduced by several different cell types, most importantly immune cells(for example monocytes, macrophages and neutrophils), but alsonon-immune cells such as fibroblasts, osteoblasts, smooth muscle cells,epithelial cells, and neurons (56). Proinflammatory cytokines contributeto various disorders, notably sepsis, through their release during aninflammatory cytokine cascade.

Inflammatory cytokine cascades contribute to deleteriouscharacteristics, including inflammation and apoptosis (32), of numerousdisorders. Included are disorders characterized by both localized andsystemic reactions, including, without limitation, diseases involvingthe gastrointestinal tract and associated tissues (such as appendicitis,peptic, gastric and duodenal ulcers, peritonitis, pancreatitis,ulcerative, pseudomembranous, acute and ischemic colitis,diverticulitis, epiglottitis, achalasia, cholangitis, coeliac disease,cholecystitis, hepatitis, Crohn's disease, enteritis, and Whipple'sdisease); systemic or local inflammatory diseases and conditions (suchas asthma, allergy, anaphylactic shock, immune complex disease, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, and sarcoidosis); diseases involving theurogential system and associated tissues (such as septic abortion,epididymitis, vaginitis, prostatitis and urethritis); diseases involvingthe respiratory system and associated tissues (such as bronchitis,emphysema, rhinitis, cystic fibrosis, adult respiratory distresssyndrome, pneumonitis, pneumoultramicroscopicsilicov-olcanoconiosis,alvealitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis);diseases arising from infection by various viruses (such as influenza,respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virusand herpes), bacteria (such as disseminated bacteremia, Dengue fever),fungi (such as candidiasis) and protozoal and multicellular parasites(such as malaria, filariasis, amebiasis, and hydatid cysts);dermatological diseases and conditions of the skin (such as burns,dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals);diseases involving the cardiovascular system and associated tissues(such as vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,congestive heart failure, periarteritis nodosa, and rheumatic fever);diseases involving the central or peripheral nervous system andassociated tissues (such as Alzheimer's disease, meningitis,encephalitis, multiple sclerosis, cerebral infarction, cerebralembolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cordinjury, paralysis, and uveitis); diseases of the bones, joints, musclesand connective tissues (such as the various arthritides and arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis, and synovitis); other autoimmune and inflammatorydisorders (such as myasthenia gravis, thryoiditis, systemic lupuserythematosus, Goodpasture's syndrome, Behcets's syndrome, allograftrejection, graft-versus-host disease, Type I diabetes, ankylosingspondylitis, Berger's disease, Type I diabetes, ankylosing spondylitis,Berger's disease, and Retier's syndrome); as well as various cancers,tumors and proliferative disorders (such as Hodgkins disease); and, inany case the inflammatory or immune host response to any primary disease(13; 55; 30; 20; 33; 25; 18; 27; 48; 24; 7; 9; 4; 3; 12; 8; 19; 15; 23;49; 34).

Mammals respond to inflammation caused by inflammatory cytokine cascadesin part though central nervous system regulation. This response has beencharacterized in detail with respect to systemic humoral responsemechanisms during inflammatory responses to endotoxin (2; 54; 21; 28).In one set of responses, afferent vagus nerve fibers are activated byendotoxin or cytokines, stimulating the release of humoralanti-inflammatory responses through glucocorticoid hormone release (51;41; 39). Previous work elucidated a role for vagus nerve signaling as acritical component in the afferent loop that modulates theadrenocorticotropin and fever responses to systemic endotoxemia andcytokinemia (14; 11; 52; 35). However, comparatively little is knownabout the role of efferent neural pathways that can modulateinflammation.

Efferent vagus nerve signaling has been implicated in facilitatinglymphocyte release from thymus via a nicotinic acetylcholine receptorresponse (1). Clinical studies have also indicated that nicotineadministration can be effective for treating some cases of inflammatorybowel disease (17; 36), and that proinflammatory cytokine levels aresignificantly decreased in the colonic mucosa of smokers withinflammatory bowel disease (40). However, none of these findings wouldsuggest that cholinergic agonists can inhibit an inflammatory cytokinecascade, particularly those mediated by macrophages. Also, there is nosuggestion in the literature that efferent vagus nerve stimulation iseffective in inhibiting these cascades.

SUMMARY OF THE INVENTION

Accordingly, the inventor has succeeded in discovering that cholinergicagonists can inhibit the release of proinflammatory cytokines from amammalian cell, either in vitro or in vivo. This inhibitory effect isuseful for inhibiting inflammatory cytokine cascades that mediate manydisease conditions. Furthermore, cholinergic agonist treatment in vivocan be effected to inhibit either local or systemic inflammatorycytokine cascades by stimulating efferent vagus nerves.

Thus, one embodiment of the present invention is directed to a method ofinhibiting the release of a proinflammatory cytokine from a mammaliancell. The method comprises treating the cell with a cholinergic agonistin an amount sufficient to decrease the amount of the proinflammatorycytokine that is released from the cell. In preferred embodiments, thecell is a macrophage. Preferably, the proinflammatory cytokine is tumornecrosis factor (TNF), interleukin (IL)-1.beta., IL-6, IL-18 or HMG-1,most preferably TNF. In preferred embodiments, the cholinergic agonistis acetylcholine, nicotine, muscarine, carbachol, galantamine,arecoline, cevimeline, or levamisole. In other preferred embodiments,the cell is in a patient suffering from, or at risk for, a conditionmediated by an inflammatory cytokine cascade, preferably appendicitis,peptic, gastric or duodenal ulcers, peritonitis, pancreatitis,ulcerative, pseudomembranous, acute or ischemic colitis, diverticulitis,epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn'sdisease, enteritis, Whipple's disease, asthma, allergy, anaphylacticshock, immune complex disease, organ ischemia, reperfusion injury, organnecrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia,hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis,septic abortion, epididymitis, vaginitis, prostatitis, urethritis,bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis,pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virusinfection, herpes infection, HIV infection, hepatitis B virus infection,hepatitis C virus infection, disseminated bacteremia, Dengue fever,candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns,dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, coeliac disease, congestive heartfailure, adult respiratory distress syndrome, Alzheimer's disease,meningitis, encephalitis, multiple sclerosis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, uveitis, arthritides, arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis,systemic lupus erythematosus, Goodpasture's syndrome, Behcets'ssyndrome, allograft rejection, graft-versus-host disease, Type Idiabetes, ankylosing spondylitis, Berger's disease, Type I diabetes,ankylosing spondylitis, Berger's disease, Retier's syndrome, or Hodgkinsdisease. In more preferred embodiments, the condition is appendicitis,peptic, gastric or duodenal ulcers, peritonitis, pancreatitis,ulcerative, pseudomembranous, acute or ischemic colitis, hepatitis,Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia,reperfusion injury, organ necrosis, hay fever, sepsis, septicemia,endotoxic shock, cachexia, septic abortion, disseminated bacteremia,burns, Alzheimer's disease, coeliac disease, congestive heart failure,adult respiratory distress syndrome, cerebral infarction, cerebralembolism, spinal cord injury, paralysis, allograft rejection orgraft-versus-host disease. In the most preferred embodiments, thecondition is endotoxic shock. In some embodiments, the cholinergicagonist treatment is effected by stimulating efferent vagus nerveactivity sufficient to inhibit the inflammatory cytokine cascade.Preferably, the efferent vagus nerve activity is stimulatedelectrically. The efferent vagus nerve can be stimulated withoutstimulating the afferent vagus nerve. Vagus nerve ganglions orpostganglionic neurons can also be stimulated. Additionally, peripheraltissues or organs that are served by the vagus nerve can also bestimulated directly.

The present invention is also directed to a method of inhibiting aninflammatory cytokine cascade in a patient. The method comprisestreating the patient with a cholinergic agonist in an amount sufficientto inhibit the inflammatory cytokine cascade, wherein the patient issuffering from, or at risk for, a condition mediated by the inflammatorycytokine cascade. The cholinergic agonist is preferably acetylcholine,nicotine, muscarine, carbachol, galantamine, arecoline, cevimeline, orlevamisole, and the condition is preferably appendicitis, peptic,gastric or duodenal ulcers, peritonitis, pancreatitis, ulcerative,pseudomembranous, acute or ischemic colitis, diverticulitis,epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn'sdisease, enteritis, Whipple's disease, asthma, allergy, anaphylacticshock, immune complex disease, organ ischemia, reperfusion injury, organnecrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia,hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis,septic abortion, epididymitis, vaginitis, prostatitis, urethritis,bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis,pneumoultramicroscopicsilicovolcanoconiosis-, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virusinfection, herpes infection, HIV infection, hepatitis B virus infection,hepatitis C virus infection, disseminated bacteremia, Dengue fever,candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns,dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliacdisease, congestive heart failure, adult respiratory distress syndrome,meningitis, encephalitis, multiple sclerosis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, uveitis, arthritides, arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis,systemic lupus erythematosus, Goodpasture's syndrome, Behcets'ssyndrome, allograft rejection, graft-versus-host disease, Type Idiabetes, ankylosing spondylitis, Berger's disease, Type I diabetes,ankylosing spondylitis, Berger's disease, Retier's syndrome, or Hodgkinsdisease. In more preferred embodiments, the condition is appendicitis,peptic, gastric or duodenal ulcers, peritonitis, pancreatitis,ulcerative, pseudomembranous, acute or ischemic colitis, hepatitis,Crohn's disease, asthma, allergy, anaphylactic shock, organ ischemia,reperfusion injury, organ necrosis, hay fever, sepsis, septicemia,endotoxic shock, cachexia, septic abortion, disseminated bacteremia,burns, Alzheimer's disease, coeliac disease, congestive heart failure,adult respiratory distress syndrome, cerebral infarction, cerebralembolism, spinal cord injury, paralysis, allograft rejection orgraft-versus-host disease. In the most preferred embodiments, thecondition is endotoxic shock. The cholinergic agonist treatment can beeffected by stimulating efferent vagus nerve activity, preferablyelectrically.

In additional embodiments, the present invention is directed to a methodfor treating a patient suffering from, or at risk for, a conditionmediated by an inflammatory cytokine cascade. The method comprisesstimulating efferent vagus nerve activity of the patient sufficient toinhibit the inflammatory cytokine cascade. Preferred methods ofstimulation and preferred conditions are as with the previouslydescribed methods.

In still other embodiments, the present invention is directed to amethod for attenuation of a systemic inflammatory response to endotoxinin a patient. The method comprises stimulating efferent vagus nerveactivity of the patient sufficient to inhibit an inflammatory cytokinecascade.

The present invention is additionally directed to a method fordetermining whether a compound is a cholinergic agonist. The methodcomprises determining whether the compound inhibits the release of aproinflammatory cytokine from a mammalian cell. In preferred embodimentsthe cell is a macrophage and the proinflammatory cytokine is TNF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph summarizing experimental results showing thatcholinergic agonists inhibit release of TNF from human macrophagecultures in a dose-dependent manner. Acetylcholine (ACh), muscarine, ornicotine was added to human macrophage cultures at the concentrationsindicated, followed by LPS addition for 4 hours. TNF concentration wasthen determined.

FIG. 2 shows autoradiographs of TNF or GADPH mRNA from LPS-stimulatedhuman macrophages treated with acetylcholine (ACh), nicotine (Nic) ormuscarine (Mus), or no cholinergic agonist, which demonstrate thatcholinergic agonists do not reduce LPS-stimulated TNF mRNA levels inmacrophages.

FIG. 3 shows micrographs of human macrophages stained with TNFantibodies demonstrating the effect of LPS and/or acetylcholine (ACh)treatment on TNF presence in the cells.

FIG. 4 is a graph summarizing experimental results showing that.alpha.-conotoxin (.alpha.-CTX), but not atropine (ATR), reverses theinhibitory effect of acetylcholine (ACh)-mediated inhibition of TNF inhuman macrophages.

FIG. 5 is a graph summarizing experimental results showing thatacetylcholine inhibits IL-1.beta. release from human macrophages in adose-dependent manner.

FIG. 6 is a graph summarizing experimental results showing thatacetylcholine inhibits L-6 release from human macrophages in adose-dependent manner.

FIG. 7 is a graph summarizing experimental results showing thatacetylcholine inhibits IL-18 release from human macrophages in adose-dependent manner.

FIG. 8 is a graph summarizing experimental results showing thatacetylcholine does not inhibit IL-10 release from human macrophages.

FIG. 9 is a graph summarizing experimental results showing that vagusnerve stimulation (STIM) after vagotomy (VGX) causes a decrease incirculating levels of TNF during endotoxemia induced by LPS.

FIG. 10 is a graph summarizing experimental results showing that vagusnerve stimulation (STIM) after vagotomy (VGX) causes a decrease inlevels of TNF in the liver during endotoxemia induced by LPS.

FIG. 11 is a graph summarizing experimental results showing that vagusnerve stimulation (STIM) after vagotomy (VGX) attenuates the developmentof hypotension (shock), as measured by mean arterial blood pressure(MABP), in rats exposed to lethal doses of endotoxin.

FIG. 12 is a graph summarizing experimental results showing that intactvagus nerve stimulation at 1V and 5V attenuates the development of shockin rats exposed to lethal doses of endotoxin.

FIG. 13 is a graph summarizing experimental results showing that intactvagus nerve stimulation at 1V and 5V causes an increase in heart rate inrats exposed to lethal doses of endotoxin.

FIG. 14 is a graph summarizing experimental results showing that intactleft vagus nerve stimulation at 1V stabilized blood pressure moreeffectively than intact right vagus nerve stimulation, in rats exposedto lethal doses of endotoxin.

FIG. 15 is a western blot and graph of experimental results showing thataddition of nicotine to RAW 264.7 macrophage-like cells inhibits theproduction of HMG-1 by the cells.

FIG. 16 is a bar graph showing the percentage of TNF in the serum ofmice injected with endotoxin and treated with mechanical stimulation ofthe vagus nerve compared with untreated controls.

FIG. 17 is a dose response curve for TNF suppression in mice injectedwith enodoxin. The y axis is the percentage of TNF in the serum relativeto untreated control; and the x axis is the number of vagus nervestimulations quantified by frequency and time.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culture, molecular biology,microbiology, cell biology, and immunology, which are well within theskill of the art. Such techniques are fully explained in the literature.See, e.g., Sambrook et al., 1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995),“Short Protocols in Molecular Biology”, John Wiley and Sons; Methods inEnzymology (several volumes); Methods in Cell Biology (several volumes),and Methods in Molecular Biology (several volumes).

The present invention is based on the discovery that treatment of aproinflammatory cytokine-producing cell with a cholinergic agonistattenuates the release of proinflammatory cytokines from that cell, andthat this attenuation process can be utilized in treatments fordisorders mediated by an inflammatory cytokine cascade (5-6). It hasfurther been discovered that stimulation of efferent vagus nerve fibersreleases sufficient acetylcholine to stop a systemic inflammatorycytokine cascade, as occurs in endotoxic shock (5), or a localizedinflammatory cytokine cascade (6). The efferent vagus nerve stimulationcan also inhibit a localized inflammatory cytokine cascade in tissuesand organs that are served by efferent vagus nerve fibers.

Accordingly, in some embodiments the present invention is directed tomethods of inhibiting the release of a proinflammatory cytokine from amammalian cell. The methods comprise treating the cell with acholinergic agonist in an amount sufficient to decrease the amount ofthe proinflammatory cytokine released from the cell.

As used herein, a cytokine is a soluble protein or peptide which isnaturally produced by mammalian cells and which act in vivo as humoralregulators at micro- to picomolar concentrations. Cytokines can, eitherunder normal or pathological conditions, modulate the functionalactivities of individual cells and tissues. A proinflammatory cytokineis a cytokine that is capable of causing any of the followingphysiological reactions associated with inflammation: vasodialation,hyperemia, increased permeability of vessels with associated edema,accumulation of granulocytes and mononuclear phagocytes, or depositionof fibrin. In some cases, the proinflammatory cytokine can also causeapoptosis, such as in chronic heart failure, where TNF has been shown tostimulate cardiomyocyte apoptosis (32; 45). Nonlimiting examples ofproinflammatory cytokines are tumor necrosis factor (TNF), interleukin(IL)-1.alpha., IL-1.beta., IL-6, IL-8, IL-18, interferon.gamma., HMG-1,platelet-activating factor (PAF), and macrophage migration inhibitoryfactor (MIF). In preferred embodiments of the invention, theproinflammatory cytokine that is inhibited by cholinergic agonisttreatment is TNF, an IL-1, IL-6 or IL-18, because these cytokines areproduced by macrophages and mediate deleterious conditions for manyimportant disorders, for example endotoxic shock, asthma, rheumatoidarthritis, inflammatory bile disease, heart failure, and allograftrejection. In most preferred embodiments, the proinflammatory cytokineis TNF.

Proinflammatory cytokines are to be distinguished from anti-inflammatorycytokines, such as IL-4, IL-10, and IL-13, which are not mediators ofinflammation. In preferred embodiments, release of anti-inflammatorycytokines is not inhibited by cholinergic agonists.

In many instances, proinflammatory cytokines are produced in aninflammatory cytokine cascade, defined herein as an in vivo release ofat least one proinflammatory cytokine in a mammal, wherein the cytokinerelease affects a physiological condition of the mammal. Thus, aninflammatory cytokine cascade is inhibited in embodiments of theinvention where proinflammatory cytokine release causes a deleteriousphysiological condition.

Any mammalian cell that produces proinflammatory cytokines are usefulfor the practice of the invention. Nonlimiting examples are monocytes,macrophages, neutrophils, epithelial cells, osteoblasts, fibroblasts,smooth muscle cells, and neurons. In preferred embodiments, the cell isa macrophage.

As used herein, a cholinergic agonist is a compound that binds to cellsexpressing cholinergic receptor activity. The skilled artisan candetermine whether any particular compound is a cholinergic agonist byany of several well known methods.

When referring to the effect of the cholinergic agonist on release ofproinflammatory cytokines or an inflammatory cytokine cascade, or theeffect of vagus nerve stimulation on an inflammatory cytokine cascade,the use of the terms “inhibit” or “decrease” encompasses at least asmall but measurable reduction in proinflammatory cytokine release. Inpreferred embodiments, the release of the proinflammatory cytokine isinhibited by at least 20% over non-treated controls; in more preferredembodiments, the inhibition is at least 50%; in still more preferredembodiments, the inhibition is at least 70%, and in the most preferredembodiments, the inhibition is at least 80%. Such reductions inproinflammatory cytokine release are capable of reducing the deleteriouseffects of an inflammatory cytokine cascade in in vivo embodiments.

Any cholinergic agonist, now known or later discovered, would beexpected to inhibit the release of proinflammatory cytokines frommammalian cells. In preferred embodiments, the cholinergic agonist isnot otherwise toxic to the cell at useful concentrations. In morepreferred embodiments, the cholinergic agonist has been usedtherapeutically in vivo or is naturally produced by mammalian cells.Nonlimiting examples include acetylcholine, nicotine, muscarine,carbachol, galantamine, arecoline, cevimeline, and levamisole. In mostpreferred in vitro embodiments, the cholinergic agonist isacetylcholine, nicotine, or muscarine. In in vivo embodiments,acetylcholine is not preferred because the compound would be expected tobe inactivated very quickly due to the widespread occurrence ofacetylcholinesterase in tissues.

The present invention is useful for studying cells in culture, forexample studying the effect of inflammatory cytokine release on thebiology of macrophages, or for testing compounds for cholinergic agonistactivity. However, in vivo applications make up many of the preferredembodiments. In those embodiments, the cell is in a patient sufferingfrom, or at risk for, a condition mediated by an inflammatory cytokinecascade. As used herein, a patient can be any mammal. However, inpreferred embodiments, the patient is a human.

The treatment of any condition mediated by an inflammatory cytokinecascade is within the scope of the invention. In preferred embodiments,the condition is one where the inflammatory cytokine cascade is effectedthrough release of proinflammatory cytokines from a macrophage. Thecondition can be one where the inflammatory cytokine cascade causes asystemic reaction, such as with septic shock. Alternatively, thecondition can be mediated by a localized inflammatory cytokine cascade,as in rheumatoid arthritis. Nonlimiting examples of conditions which canbe usefully treated using the present invention include those conditionsenumerated in the background section of this specification. Preferably,the condition is appendicitis, peptic, gastric or duodenal ulcers,peritonitis, pancreatitis, ulcerative, pseudomembranous, acute orischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis,cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease,asthma, allergy, anaphylactic shock, immune complex disease, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis,vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis,cystic fibrosis, pneumonitis,pneumoultramicroscopicsilicovolcanoconiosis-, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytialvirus, herpes, disseminated bacteremia, Dengue fever, candidiasis,malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis,dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis,endocarditis, arteritis, atherosclerosis, thrombophlebitis,pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa,rheumatic fever, Alzheimer's disease, coeliac disease, congestive heartfailure, adult respiratory distress syndrome, meningitis, encephalitis,multiple sclerosis, cerebral infarction, cerebral embolism,Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury,paralysis, uveitis, arthritides, arthralgias, osteomyelitis, fasciitis,Paget's disease, gout, periodontal disease, rheumatoid arthritis,synovitis, myasthenia gravis, thryoiditis, systemic lupus erythematosus,Goodpasture's syndrome, Behcets's syndrome, allograft rejection,graft-versus-host disease, Type I diabetes, ankylosing spondylitis,Berger's disease, Type I diabetes, ankylosing spondylitis, Berger'sdisease, Retier's syndrome, or Hodgkins disease. In more preferredembodiments, the condition is appendicitis, peptic, gastric or duodenalulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acuteor ischemic colitis, hepatitis, Crohn's disease, asthma, allergy,anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis,hay fever, sepsis, septicemia, endotoxic shock, cachexia, septicabortion, disseminated bacteremia, burns, Alzheimer's disease, coeliacdisease, congestive heart failure, adult respiratory distress syndrome,cerebral infarction, cerebral embolism, spinal cord injury, paralysis,allograft rejection or graft-versus-host disease. In the most preferredembodiments, the condition is endotoxic shock.

The route of administration of the cholinergic agonist depends on thecondition to be treated. For example, intravenous injection may bepreferred for treatment of a systemic disorder such as septic shock, andoral administration may be preferred to treat a gastrointestinaldisorder such as a gastric ulcer. The route of administration and thedosage of the cholinergic agonist to be administered can be determinedby the skilled artisan without undue experimentation in conjunction withstandard dose-response studies. Relevant circumstances to be consideredin making those determinations include the condition or conditions to betreated, the choice of composition to be administered, the age, weight,and response of the individual patient, and the severity of thepatient's symptoms. Thus, depending on the condition, the cholinergicagonist can be administered orally, parenterally, intranasally,vaginally, rectally, lingually, sublingually, bucally, intrabuccaly andtransdermally to the patient.

Accordingly, cholinergic agonist compositions designed for oral,lingual, sublingual, buccal and intrabuccal administration can be madewithout undue experimentation by means well known in the art, forexample with an inert diluent or with an edible carrier. Thecompositions may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thepharmaceutical compositions of the present invention may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

Cholinergic agonist compositions of the present invention can easily beadministered parenterally such as for example, by intravenous,intramuscular, intrathecal or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the cholinergicagonist compositions of the present invention into a solution orsuspension. Such solutions or suspensions may also include sterilediluents such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents. Parenteral formulations may also include antibacterial agentssuch as for example, benzyl alcohol or methyl parabens, antioxidantssuch as for example, ascorbic acid or sodium bisulfite and chelatingagents such as EDTA. Buffers such as acetates, citrates or phosphatesand agents for the adjustment of tonicity such as sodium chloride ordextrose may also be added. The parenteral preparation can be enclosedin ampules, disposable syringes or multiple dose vials made of glass orplastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120.degree.C., dissolving the cholinergic agonist in the glycerin, mixing theheated glycerin after which purified water may be added, and pouring thehot mixture into a suppository mold.

Transdermal administration includes percutaneous absorption of thecholinergic agonist through the skin. Transdermal formulations includepatches (such as the well-known nicotine patch), ointments, creams,gels, salves and the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the cholinergic agonist. As usedherein, nasally administering or nasal administration includesadministering the cholinergic agonist to the mucous membranes of thenasal passage or nasal cavity of the patient. As used herein,pharmaceutical compositions for nasal administration of a cholinergicagonist include therapeutically effective amounts of the agonistprepared by well-known methods to be administered, for example, as anasal spray, nasal drop, suspension, gel, ointment, cream or powder.Administration of the cholinergic agonist may also take place using anasal tampon or nasal sponge.

In accordance with the present invention, it has also been discoveredthat the cholinergic agonist can be administered to the patient in theform of acetylcholine by stimulating efferent vagus nerve fibers. As iswell known, efferent vagus nerve fibers secrete acetylcholine uponstimulation. Such stimulation releases sufficient acetylcholine to beeffective in inhibiting a systemic inflammatory cytokine cascade as wellas a localized inflammatory cytokine cascade in a tissue or organ thatis served by efferent branches of the vagus nerve, including thepharynx, the larynx, the esophagus, the heart, the lungs, the stomach,the pancreas, the spleen, the kidneys, the adrenal glands, the small andlarge intestine, the colon, and the liver.

The effect of vagus nerve stimulation on the inhibition of inflammatorycytokine cascades is not necessarily limited to that caused byacetylcholine release. The scope of the invention also encompasses anyother mechanism that is partly or wholly responsible for the inhibitionof inflammatory cytokine cascades by vagus nerve stimulation.Nonlimiting examples include the release of serotonin agonists orstimulation of other neurotransmitters.

As used herein, the vagus nerve is used in its broadest sense, andincludes any nerves that branch off from the main vagus nerve, as wellas ganglions or postganglionic neurons that are connected to the vagusnerve. The vagus nerve is also known in the art as the parasympatheticnervous system and its branches, and the cholinergic nerve.

The efferent vagus nerve fibers can be stimulated by any means.Nonlimiting examples include: mechanical means such as a needle,ultrasound, or vibration. Mechanical stimulation can also be carried outby carotid massage, oculocardiac reflex, dive reflex and valsalvamaneuver. Specific examples where an inflammatory response was reduce bymechanical vagal nerve stimulation are provided in Examples 5 and 6. Theefferent vagal nerve fibers can also be stimulate by electromagneticradiation such as infrared, visible or ultraviolet light; heat, or anyother energy source. In preferred embodiments, the vagus nerve isstimulated electrically, using for example a commercial vagus nervestimulator such as the Cyberonics NCP®, or an electric probe. Theefferent vagus nerve can be stimulated by stimulating the entire vagusnerve (i.e., both the afferent and efferent nerves), or by isolatingefferent nerves and stimulating them directly. The latter method can beaccomplished by separating the afferent from the efferent fibers in anarea of the nerve where both types of fibers are present. Alternatively,the efferent fiber is stimulated where no afferent fibers are present,for example close to the target organ served by the efferent fibers. Theefferent fibers can also be stimulated by stimulating the target organdirectly, e.g., electrically, thus stimulating the efferent fibers thatserve that organ. In other embodiments, the ganglion or postganglionicneurons of the vagus nerve can be stimulated. The vagus nerve can alsobe cut and the distal end can be stimulated, thus only stimulatingefferent vagus nerve fibers (see, e.g., Example 2).

The amount of stimulation useful to inhibit an inflammatory cytokinecascade can be determined by the skilled artisan without undueexperimentation for any condition to be treated. To inhibit a systemicinflammatory cytokine cascade, as induced with endotoxin, constantvoltage stimuli of 1 to 5 V, at 2 ms and 1 Hz, for 10 min. beforeexposure and 10 min. after exposure, will inhibit the systemicinflammatory cytokine cascade sufficiently to prevent death of thesubject by endotoxic shock (see Examples 2 and 3).

In other embodiments, the invention is directed to methods of inhibitingan inflammatory cytokine cascade in a patient. The methods comprisetreating the patient with a cholinergic agonist in an amount sufficientto inhibit the inflammatory cytokine cascade. In preferred embodiments,the patient is suffering from, or at risk for, a condition mediated bythe inflammatory cytokine cascade.

Cholinergic agonists useful for these embodiments have been previouslydiscussed and include acetylcholine, nicotine, muscarine, carbachol,galantamine, arecoline, cevimeline, and levamisole. Also as previouslydiscussed, acetylcholine can be administered by stimulating efferentvagus nerve fibers.

In additional embodiments, the present invention is directed to a methodfor treating a patient suffering from, or at risk for, a conditionmediated by an inflammatory cytokine cascade. The method comprisesstimulating efferent vagus nerve activity sufficient to inhibit theinflammatory cytokine cascade. Methods for stimulating efferent vagusnerve fibers have been previously discussed.

The present invention is also directed to methods for determiningwhether a compound is a cholinergic agonist. The method comprisesdetermining whether the compound inhibits the release of aproinflammatory cytokine from a mammalian cell.

For this method, the cell can be any cell that can be induced to producea proinflammatory cytokine. In preferred embodiments, the cell is animmune cell, for example macrophages, monocytes, or neutrophils. In themost preferred embodiments, the cell is a macrophage.

The proinflammatory cytokine to be measured for inhibition can be anyproinflammatory cytokine that can be induced to be released from thecell. In preferred embodiments, the cytokine is TNF. Evaluation of theinhibition of cytokine production can be by any means known, includingquantitation of the cytokine (e.g., with ELISA), or by bioassay, (e.g.determining whether proinflammatory cytokine activity is reduced).

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1 Cholinergic Agonists Inhibit Release of ProinflammatoryCytokines from Macrophages

1. Materials and Methods

Human macrophage cultures were prepared as follows. Buffy coats werecollected from the blood of healthy individual donors to the Long IslandBlood Bank Services (Melville, N.Y.). Primary blood mononuclear cellswere isolated by density-gradient centrifugation through Ficoll/Hypaque(Pharmacia, N.J.), suspended (8.times.10.sup.6 cells/ml) in RPMI 1640medium supplemented with 10% heat inactivated human serum (GeminiBio-Products, Inc., Calabasas, Calif.), and seeded in flasks (PRIMARIA;Beckton and Dickinson Labware, Franklin Lakes, N.J.). After incubationfor 2 hours at 37.degree. C., adherent cells were washed extensively,treated briefly with 10 mM EDTA, detached, resuspended (10.sup.6cells/ml) in RPMI medium (10% human serum), supplemented with humanmacrophage colony stimulating factor (MCSF; Sigma Chemical Co., St.Louis, Mo.; 2 ng/ml), and seeded onto 24-well tissue culture plates(PRIMARIA; Falcon) (10.sup.6 cells/well). Cells were allowed todifferentiate for 7 days in the presence of MCSF. On day 7 the cellswere washed 3 times with 1.times. Dulbecco's phosphate buffered saline(PBS, GibcoBRL, Life Technologies, Rockville, Md.), fresh medium devoidof MCSF was added, and experiments performed as indicated.

RNase protection assays were performed as follows. Total RNA wasisolated from cultured cells by using TRIzol reagent (GIBCO BRL,Rockville, Md.) following the manufacturer's instructions, andelectrophoresed on 1.2% agarose/17% formaldehyde gel for verification ofthe integrity of the RNA samples. The RNase protection assay wasconducted using a kit obtained from PharMingen (San Diego, Calif.). Theanti-sense RNA probe set (hck-3) was labeled with [a-.sup.32P] UTP(Sp.Act. 800 Ci/mmol, Amersham, Arlington Heights, Ill.) using T7 RNApolymerase. Molecular weight markers were prepared by using pBR-322plasmid DNA digested with MSP I (New England Bio Labs, Beverly, Mass.)and end-labeled using [a-.sup.32P] dCTP (Sp. Act. 800 Ci/mmol, Amersham,Arlington Heights, Ill.) with Klenow enzyme (Strategene, La Jolla,Calif.).

TNF immunohistochemistry was performed as follows. Human macrophageswere differentiated as described above, and grown on glass chamberslides (Nunc, Naperville, Ill.). Slides were incubated in a blockingsolution (1% BSA, 5% normal goat serum, 0.3% Triton X-100 in PBS) for 1hour at room temperature and then incubated for 24 hours at 4.degree. C.with a primary mouse anti-human TNF monoclonal antibody (Genzyme,Cambridge, Mass.) diluted 1:100 in PBS containing 0.3% Triton X-100,0.1% BSA, and 3% normal goat serum. Washed sections were incubated for 2hours with secondary biotinylated anti-mouse IgG (1:200, VectorLaboratories, Inc., Burlingame, Calif.). The reaction product wasvisualized with 0.003% hydrogen peroxide and 0.05% 3,3′-diaminobenzidinetetrahydrochloride as a chromogen. Negative controls were incubated inthe absence of primary antibodies (not shown). Slides were analyzed on alight microscope (Olympus BX60, Japan) using a MetaMorth Imaging System(Universal Imaging Co., West Chester, Pa.).

2. Results

Primary human macrophage cultures were established by incubating humanperipheral blood mononuclear cells in the presence of macrophage colonystimulating factor (MCSF; Sigma Chemical Co., St. Louis, Mo.). Thesecells were used in experiments to determine the effects of cholinergicagonists on TNF levels in macrophage cultures conditioned by exposure toLPS for 4 hours (FIG. 1). In those experiments, acetylcholine chloride(ACh; Sigma Chemical Co., St. Louis, Mo.) was added to human macrophagecultures at the indicated concentrations (squares) in the presence ofthe acetylcholinesterase inhibitor pyridostigmine bromide (1 mM, SigmaChemical Co., St. Louis, Mo.). Muscarine (triangles) and nicotine(circles) (Sigma Chemical Co., St. Louis, Mo.) were added in theconcentrations indicated (FIG. 1). LPS was added five minutes later (100ng/ml), and conditioned supernatants collected after 4 hours ofstimulation for subsequent analysis by TNF enzyme-linked immunosorbentassay (ELISA). All the experimental conditions were performed intriplicate. Data from nine separate macrophage preparations are shown asMean.+−.SEM; n=9.

As shown in FIG. 1, acetylcholine, nicotine, and muscarine all inhibitedTNF release in a dose dependent manner. Comparable inhibition of TNFrelease by acetylcholine was observed in macrophage culture mediaconditioned by exposure to LPS for 20 hours (not shown), indicating thatthe inhibitory effect of acetylcholine on TNF did not merely delay theonset of the TNF response. Inhibition of TNF was also observed inmacrophage cultures treated with carbachol, a chemically distinctcholinergic agonist (not shown).

The molecular mechanism of TNF inhibition was investigated by measuringTNF mRNA levels in an RNase protection assay. In those experiments (FIG.2), macrophages were incubated in the presence of ACh (100 .mu.M),muscarine (Mus, 100.mu.M), nicotine (Nic, 100 .mu.M) or medium alone for5 minutes followed by 2 hour exposure to LPS (100 ng/ml). ACh was addedwith pyridostigmine bromide (1 mM). Control wells were incubated withmedium alone for 2 hours. Expression of the GAPDH gene product wasmeasured to control for mRNA loading.

TNF mRNA levels in acetylcholine-treated, LPS-stimulated macrophages didnot decrease as compared to vehicle-treated, LPS-stimulated macrophages,even when acetylcholine was added in concentrations that inhibited TNFprotein release (FIG. 2). This indicates that acetylcholine suppressesTNF release through a post-transcriptional mechanism.

To determine whether acetylcholine inhibited macrophage TNF synthesis ormacrophage TNF release, monoclonal anti-TNF antibodies were used tolabel cell-associated TNF in human macrophage cultures. In thoseexperiments (FIG. 3), cells were exposed to either ACh (100 .mu.M),either alone or in the presence of pyridostigmine bromide (1 mM), fiveminutes before LPS (100 ng/ml) treatment. Two hours later the cells werefixed in buffered 10% formalin and subjected to immunocytochemicalanalysis using primary mouse anti-hTNF monoclonal antibodies asdescribed in Materials and Methods.

Those experiments established that acetylcholine significantlyattenuated the appearance of LPS-stimulated TNF immunoreactivity inmacrophages (FIG. 3). Considered together, these results indicate thatthe inhibitory effect of acetylcholine on human macrophage TNFproduction occurs through the post-transcriptional suppression of TNFprotein synthesis, or possibly through an increased rate of degradationof intracellular TNF (FIG. 3).

Previous work indicated that peripheral blood mononuclear cells expressnicotinic and muscarinic acetylcholine receptors (37-38; 53). To definepharmacologically the type of macrophage cholinergic receptor activitiesinvolved in modulating the TNF response, the results in FIG. 1 werefurther analyzed. Nicotine significantly inhibited TNF release in adose-dependent manner; the effective concentration of nicotine thatinhibited 50% of the TNF response (E.C..sub.50) was estimated to be8.3.+−.7.1 nM (n=9). This E.C..sub.50 for nicotine compared favorablywith the E.C..sub.50 for acetylcholine-mediated inhibition of TNF(acetylcholine E.C..sub.50=20.2.+−.8.7 nM, n=9). Muscarine alsosignificantly inhibited TNF release, although it was a much lesseffective inhibitor of macrophage TNF as compared to eitheracetylcholine or nicotine (muscarine E.C..sub.50=42.4.+−.18.6 mM, n=9;P<0.01 vs nicotine or acetylcholine).

To establish whether acetylcholine inhibited TNF primarily through theactivity of nicotinic or muscarinic acetylcholine receptors, thespecific muscarinic antagonist, atropine, was added to LPS-stimulatedmacrophage cultures that were co-treated with acetylcholine (FIG. 4). Ialso addressed whether the nicotinic acetylcholine receptor activitythat mediated inhibition of TNF was a-bungarotoxin-sensitive ora-bungarotoxin-insensitive (FIG. 4). Conditions for macrophage cultureand TNF assays were as previously described. Atropine (striped bars) (1mM; Sigma Chemical Co., St. Louis, Mo.) or .alpha.-conotoxin (blackbars) (0.1, 0.01 mM; Oncogene Research Products, Cambridge, Mass.) wereadded to macrophage cultures 5 minutes prior to acetylcholine (10 .mu.M)and LPS (100 ng/ml). Data shown are Mean.+−.SEM of 3 separateexperiments using different macrophages prepared from separate donors.

Addition of atropine, even in concentrations as high as 1 mM, failed torestore TNF release in acetylcholine-treated macrophage cultures (FIG.4). Note that Acetylcholine inhibited TNF release by 80%, but this wasnot reversed by atropine. However, addition of .alpha.-conotoxin toacetylcholine-treated LPS-stimulated macrophage cultures significantlyreversed the inhibitory effect of acetylcholine in a dose dependentmanner (FIG. 4). (**P<0.005 vs ACh; *P<0.05 vs ACh). Neither atropinenor .alpha.-conotoxin altered TNF production in vehicle-treated cultures(not shown). Considered together, these data provide evidence that theinhibitory effect of acetylcholine on the LPS-induced TNF response inhuman macrophage cultures is mediated primarily by.alpha.-bungarotoxin-sensitive, nicotinic acetylcholine receptors.Acetylcholine levels in mammalian tissues can reach the millimolar range(50); however so, it is possible that both the nicotinic and muscarinicmacrophage acetylcholine receptor activities described here participatein the inhibition of macrophage TNF synthesis in vivo.

To assess specificity, the release of other macrophage-derived cytokineswas measured in LPS-stimulated macrophage cultures treated withacetylcholine. In those experiments, human macrophage cultures wereincubated with ACh at the indicated concentrations in the presence ofpyridostigmine bromide (1 mM) and LPS (100 ng/ml) for 20 hours.IL-1.beta. (FIG. 5), IL-6 (FIG. 6) and IL-10 (FIG. 8) levels weremeasured in media using commercially available ELISA kits (R&D SystemsInc., Minneapolis, Minn.). IL-18 (FIG. 7) levels were determined byspecific ELISA (Medical & Biological Laboratories Co., Ltd., Nagoya,Japan). Each sample was analyzed in triplicate. Data are expressed asMean.+−.SEM from 4 separate experiments using macrophages prepared from4 separate healthy donors. These experiments established thatacetylcholine dose-dependently inhibits the release of otherLPS-inducible cytokines (IL-1.beta., IL-6 and IL-18, FIGS. 5, 6, and 7,respectively), but does not prevent the constitutive release of theanti-inflammatory cytokine IL-10 (FIG. 8). Thus, acetylcholinespecifically inhibits release of pro-inflammatory cytokines (FIGS. 5-7)by LPS-stimulated human macrophage cultures, but does not suppressrelease of the anti-inflammatory cytokine IL-10 (FIG. 8). Staining withtetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Chemical Co., St. Louis, Mo.), and Trypanblue exclusion of macrophage cultures treated with LPS and acetylcholineindicated that specific LPS-inducible cytokine inhibition was not due tocytotoxicity (not shown).

The molecular mechanism of acetylcholine inhibition of IL-1.beta. andIL-6 was investigated further by measuring gene-specific mRNA levelswith biotin-labeled capture oligonucleotide probes in a calorimetricmicroplate assay (Quantikine mRNA, R&D Systems, Inc., Minneapolis,Minn.). Stimulation of human macrophage cultures with LPS for 2 hourssignificantly increased the mRNA levels of IL-1.beta. as compared tovehicle-treated controls (vehicle-treated IL-1.beta. mRNA=120.+−.54attomole/ml vs LPS-stimulated IL-1.beta. mRNA=1974.+−.179 attomole/ml;n=3; P<0.01). Addition of acetylcholine in concentrations (100 nM) thatsignificantly inhibited IL-1.beta. protein release did not significantlyalter macrophage IL-1.beta. mRNA levels (acetylcholine-treatedLPS-stimulated IL-1.beta. mRNA=2128.+−.65 attomole/ml; n=3). Similarly,LPS-stimulated IL-6 mRNA levels in macrophages were not significantlyaltered by acetylcholine concentrations that significantly inhibitedIL-6 protein (LPS-stimulated IL-6 mRNA=1716.+−.157 attomole/ml vs.acetylcholine-treated LPS-stimulated IL-6 mRNA=1872.+−.91 attomole/ml;n=3). Together, these observations give evidence that acetylcholinepost-transcriptionally inhibits the LPS-stimulated release of TNF,IL-1.beta. and IL-6 in macrophages.

The present results indicate that differentiated human macrophagecultures are extremely sensitive to acetylcholine and nicotine. Previousreports of cholinergic receptor activity in human peripheral bloodmononuclear cells that were not differentiated into macrophages (53; 29;46) suggested that maximal cholinergic responses required micromolarconcentrations of cholinergic agonists. Our own studies establish thatsignificantly higher concentrations of acetylcholine are required tosuppress cytokine synthesis in differentiated human macrophages(acetylcholine E.C..sub.50 for inhibiting TNF=0.8.+−.0.2 mM, n=3). Thepharmacological results now implicate an .alpha.-bungarotoxin-sensitive,nicotinic acetylcholine receptor activity that can modulate themacrophage cytokine response. This type of cholinergic receptor activityis similar to that previously described in peripheral blood mononuclearcells (53), except that macrophages are significantly more sensitive tocholinergic agonists as compared to peripheral blood mononuclear cells.The skilled artisan would not necessarily have expected macrophages tobe so sensitive to cholinergic agonists, or even have any sensitivity atall, given what was previously known with mononuclear cells. Indeed,recent evidence in our lab has revealed nicotinic receptor subunitexpression patterns in macrophages that are distinct from monocytes.Therefore, the skilled artisan would understand that moleculardifferences underlie the greater sensitivity to cholinergic agonists ofmacrophages over monocytes.

EXAMPLE 2 Inhibition of Endotoxic Shock by Stimulation of Efferent VagusNerve Fibers

To determine whether direct stimulation of efferent vagus nerve activitymight suppress the systemic inflammatory response to endotoxin, adultmale Lewis rats were subjected to bilateral cervical vagotomy, or acomparable sham surgical procedure in which the vagus nerve was isolatedbut not transected. Efferent vagus nerve activity was stimulated invagotomized rats by application of constant voltage stimuli to thedistal end of the divided vagus nerve 10 min before and again 10 minafter the administration of a lethal LPS dose (15 mg/kg, i.v.). Ananimal model of endotoxic shock was utilized in these experiments. Adultmale Lewis rats (280-300 g, Charles River Laboratories, Wilmington,Mass.) were housed at 22.degree. C. on a 12 h light/dark cycle. Allanimal experiments were performed in accordance with the NationalInstitute of Health Guidelines under the protocols approved by theInstitutional Animal Care and Use Committee of North Shore UniversityHospital/New York University School of Medicine. Rats were anesthetizedwith urethane (1 g/kg, intraperitoneally), and the trachea, the commoncarotid artery, and the jugular vein were cannulated with polyethylenetubing (Clay Adams, Parsippany, N.J.). The catheter implanted into theright common carotid artery was connected to a blood pressure transducerand an Acquisition System (MP 100, BIOPAC Systems, Inc., Santa Barbara,Calif.) for continuous registration of mean arterial blood pressure(MABP in FIG. 9). Animals were subjected to bilateral cervical vagotomy(VGX, n=7) alone or with electrical stimulation (VGX+STIM, n=7) or shamsurgery (SHAM, n=7). In vagotomized animals, following a ventralcervical midline incision, both vagus trunks were exposed, ligated witha 4-0 silk suture, and divided. In sham-operated animals both vagaltrunks were exposed and isolated from the surrounding tissue but nottransected. Electrical stimulation of the vagus nerve was performed inanimals previously subjected to vagotomy. In these groups, the distalend of right vagus nerve trunk was placed across bipolar platinumelectrodes (Plastics One Inc., Roanoke, Va.) connected to a stimulationmodule (STM100A, Harvard Apparatus, Inc., Holliston, Miss.) ascontrolled by an Acquisition System (MP100, BIOPAC Systems, Inc., SantaBarbara, Calif.). Constant voltage stimuli (5 V, 2 ms, 1 Hz) wereapplied to the nerve for 20 min (10 min before LPS administration and 10min after). Lipopolysaccharide (Escherichia coli 0111:B4; Sigma ChemicalCo, St. Louis, Mo.; 10 mg/ml in saline) was sonicated for 30 minutes,and administered at a lethal dose (15 mg/kg, i.v.). Blood was collectedfrom the right carotid artery 1 hour after LPS administration. Serum TNFlevels were quantified by the L929 bioactivity assay. To determine liverTNF levels, animals were euthanized and livers rapidly excised, rinsedof blood, homogenized by polytron (Brinkman, Westbury, N.Y.) inhomogenization buffer (PBS, containing 0.05% sodium azide, 0.5% TritonX-100 and a protease inhibitor cocktail (2 tablets/10 ml PBS, BoehringerMannheim, Germany); pH 7.2; 4.degree. C.), and then sonicated for 10minutes. Homogenates were centrifuged at 12,000 g for 10 minutes, andTNF levels in supernatants determined by ELISA (Biosource International,Camarillo, Calif.). Protein concentrations in the supernatants weremeasured by the Bio-Rad protein assay (Bio-Rad Lab., Hercules, Calif.),and liver TNF content normalized by the amount of protein in the sample.Blood samples were collected 1 hour after LPS and TNF was measured byL929 assay. *−P<0.05,**−P<0.005 vs. SHAM+LPS,#−P<0.05 vs. VGX+LPS.

As shown in FIG. 9, the results establish that electrical stimulation ofthe efferent vagus nerve significantly attenuates peak serum TNF levels;vagotomy without electrical stimulation significantly increased peakserum TNF levels as compared to sham-operated controls (P<0.05).

TNF levels in liver homogenates were measured next, because liver is aprinciple source of peak serum TNF during endotoxemia (26; 16).Electrical stimulation of the distal vagus nerve significantlyattenuated hepatic TNF synthesis as compared to sham-operated controls(FIG. 10). In that figure, *−P<0.05 vs. SHAM+LPS,#−P<0.05 vs. VGX+LPS.These data directly implicate efferent vagus nerve signaling in theregulation of TNF production in vivo.

It was theoretically possible that electrical stimulation of the vagusnerve induced the release of humoral anti-inflammatory hormones orcytokines that inhibit TNF production. Measurements of corticosteroneand IL-10 levels in sham-operated controls were performed (Table 1) todetermine this.

In those studies, animals were subjected to either sham surgery (SHAM),vagotomy (VGX), or electrical stimulation with vagotomy (VGX+STIM) 30minutes before systemic administration of LPS (15 mg/kg). Blood sampleswere collected 1 hour after administration of LPS or vehicle. Serumcorticosterone was measured by radioimmunoassay (ICN Biomedicals, CostaMesa, Calif.) and IL-10 was determined by ELISA (BioSourceInternational, Camarillo, Calif.). All assays were performed intriplicate. The results are shown in Table 1, which indicates thatendotoxemia was associated with increases in corticosterone and IL-10levels. In agreement with previous studies, vagotomy significantlyreduced corticosterone levels, in part because it eliminated theafferent vagus nerve signals to the brain that are required for asubsequent activation of the hypothalamic-pituitary-adrenal axis (14;11). This decreased corticosteroid response and likely contributed tothe increased levels of TNF observed in the serum and liver ofvagotomized animals (FIGS. 9 and 10), because corticosteroids normallydown-regulate TNF production (41; 39). Direct electrical stimulation ofthe peripheral vagus nerve did not stimulate an increase in either thecorticosteroid or the IL-10 responses. Thus, suppressed TNF synthesis inthe serum and liver after vagus nerve stimulation could not beattributed to the activity of these humoral anti-inflammatory mediators.

TABLE 1 Effects of vagotomy and vagus nerve stimulation on serum IL-10and corticosteroid levels during lethal endotoxemia. Group of animalsIL-10 (ng/ml) Corticosterone (ng/ml) SHAM+vehicle N.D. 160.+−.20SHAM+LPS 8.+−.0.3 850.+−.50 Vagotomy+LPS 9.+−.0.4 570.+−.34*Vagotomy+LPS+Stimulation 9.+−.0.5 560.+−. 43*

Data shown are Mean.+−.SEM, n=7 animals per group. *p<0.05 vs. SHAM+LPS.

FIG. 11 shows the results of measurement of mean arterial blood pressure(MABP) in the same groups of animals as in FIG. 9 and 10 (as describedin methods). Circles—sham-operated rats (SHAM), triangles—vagotomizedrats (VGX), squares—animals with electrical stimulation of the vagusnerve and vagotomy (VGX+STIM). LPS (15 mg/kg, i.v.) was injected attime=0. All data are expressed as % of MABP [MABP/MABP (attime=0).times.100%], Mean.+−.SEM; n=7. Sham-surgery, vagotomy andelectrical stimulation with vagotomy did not significantly affect MABPin vehicle-treated controls (not shown).

Peripheral vagus nerve stimulation significantly attenuated thedevelopment of LPS-induced hypotension (shock) in rats exposed to lethaldoses of endotoxin (FIG. 11). This observation was not unexpected,because TNF is a principle early mediator of acute endotoxin-inducedshock (43-44). Vagotomy alone (without electrical stimulation)significantly shortened the time to development of shock as compared tosham-operated controls (sham time to 50% drop in mean arterial bloodpressure 30.+−.3 minutes versus vagotomy time to 50% drop in meanarterial blood pressure=15.+−.2 minutes; P<0.05). This amplifieddevelopment of shock following vagotomy alone corresponded to thedecreased corticosteroid response and the increased TNF response.

Acetylcholine is a vasodilator that mediates nitric oxide-dependentrelaxation of resistance blood vessels which causes a decrease in bloodpressure. Thus, we wished to exclude the possibility that stimulation ofthe efferent vagus might have mediated a paradoxical hypertensiveresponse. Hypertension was not observed following vagus nervestimulation of controls given saline instead of endotoxin (not shown),indicating that protection against endotoxic shock by vagus nervestimulation is specific. Considered together, these observationsindicate that stimulation of efferent vagus nerve activity downregulatessystemic TNF production and the development of shock during lethalendotoxemia.

EXAMPLE 3 Stimulation of Intact Vagus Nerve Attenuates Endotoxic Shock

Experiments were conducted to determine whether the inhibition ofinflammatory cytokine cascades by efferent vagus nerve stimulation iseffective by stimulation of an intact vagus nerve. Stimulation of leftand right vagus nerves were also compared.

The vagus nerves of anesthetized rats were exposed, and the left commoniliac arteries were cannulated to monitor blood pressure and heart rate.Endotoxin (E. coli 0111:B4; Sigma) was administered at a lethal dose (60mg/kg). In treated animals, either the left or the right intact vagusnerve was stimulated with constant voltage (5V or 1V, 2 ms, 1 Hz) for atotal of 20 min., beginning 10 min. before and continuing 10 min. afterLPS injection. Blood pressure and heart rate were through the use of aBio-Pac M100 computer-assisted acquisition system. FIGS. 12-14 show theresults of these experiments.

As shown in FIG. 12, within minutes after LPS injection, the bloodpressure began to decline in both unstimulated rats and rats treatedwith a low dose (1V) of vagus nerve stimulation, while rats treated witha high dose (5V) of stimulation maintained more stable blood pressures.Between 30 and 40 min. post-LPS, the blood pressure stabilized inanimals treated with a low dose of voltage.

FIG. 13 shows the heart rate of the experimental animals. Within minutesafter LPS injection, the heart rate began to increase in rats stimulatedwith a high dose (5V) of vagus nerve stimulation. On the other hand, theheart rates of both unstimulated rats and rats stimulated with a lowdose (1V) of voltage remained stable for approximately 60 min. post-LPS.After one hour, the heart rates of the rats treated with a low dose ofstimulation began to increase, and reached levels comparable to thoserats receiving a high dose of vagus nerve stimulation.

FIG. 14 compares left vs. right vagus nerve stimulation. Endotoxicanimals were treated with 1V stimulation in either the left or the rightvagus nerve. Within minutes after LPS injection, the blood pressurebegan to decline in all three stes of animals (unstimulated, leftstimulation, right stimulation). Though both sets of stimulated animalsrecovered blood pressure, those animals receiving stimulation in theleft vagus nerve maintained more stable blood pressures for the durationof the experiment. However, the difference in results between left andright vagus nerve stimulation was not statistically significant, andwould not be expected to have any practical difference.

This set of experiments confirms that stimulation of an intact vagusnerve can effectively inhibit an inflammatory cytokine cascadesufficiently to alleviate conditions caused by the cascade.

EXAMPLE 4 Inhibition of HMG-1 Release from Macrophages by Nicotine

Experiments were performed to determine whether the inhibitory effect ofcholinergic agonists on proinflammatory cytokines applied to HMG-1.Murine RAW 264.7 macrophage-like cells (American Type CultureCollection, Rockville, Md., USA) were grown in culture under DMEMsupplemented with 10% fetal bovine serum and 1% glutamine. When thecells were 70-80% confluent, the medium was replaced by serum-freeOPTI-MEM 1 medium. Nicotine (Sigma) was then added at 0, 0.1, 1, 10 or100 .mu.M. Five minutes after adding the nicotine, the cultures weretreated with LPS (500 ng/ml). Culture medium was collected after 20 hr.The culture medium was concentrated with a Centricon T 10 filter, thenanalyzed by western blot, using an anti-HMG-1 polyclonal antisera (WO00/47104) and standard methods. Band densities were determined using aBio-Rad Imaging densitometer.

The results are shown in FIG. 15. The HMG-1 bands are shown along thetop, with the corresponding nicotine and LPS concentrations, and thedensities of the bands shown are graphed in the graph below. FIG. 15clearly shows that nicotine inhibited HMG-1 production in adose-dependent manner. This demonstrates that HMG-1 behaves as aproinflammatory cytokine in that its production can be inhibited by acholinergic agonist.

The neural-immune interaction described here, which we term the“cholinergic anti-inflammatory pathway,” can directly modulate thesystemic response to pathogenic invasion. The observation thatparasympathetic nervous system activity influences circulating TNFlevels and the shock response to endotoxemia has widespreadimplications, because it represents a previously unrecognized, direct,and rapid endogenous mechanism that can be activated to suppress thelethal effects of biological toxins. The cholinergic anti-inflammatorypathway is positioned to function under much shorter response times ascompared to the previously described humoral anti-inflammatory pathways.Moreover, activation of parasympathetic efferents during systemicstress, or the “flight or fight” response, confers an additionalprotective advantage to the host by restraining the magnitude of apotentially lethal peripheral immune response.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

EXAMPLE 5 Mechanical Vagus Nerve Stimulation is Sufficient to InhibitInflammatory Cytokine Release

To determine the activation sensitivity of the cholinergicanti-inflammatory via VNS, the ability of mechanical nerve stimulationto activate the cholinergic anti-inflammatory pathway was examined. Male8- to 12-week-old BALB/c mice (25-30 g; Taconic) were housed at 25° C.on a 12 h light/dark cycle. Animals were allowed to acclimate to thefacility for at least 7 days prior to experimental manipulation.Standard mouse chow and water were freely available. All animalexperiments were performed in accordance with the National Institutes ofHealth (NIH) Guidelines under protocols approved by the InstitutionalAnimal Care and Use Committee of the North Shore-Long Island JewishResearch Institute.

Mice were anesthetized with isoflurane (1.5-2.0%) and placed supine onthe operating table. A ventral cervical midline incision was used toexpose and isolate the left cervical vagus nerve. The left vagus nervewas exposed via a midline cervical incision. After isolating the nervefrom the surrounding structures, the surgery was terminated, withoutsubsequent electrode placement. LPS administration preceded surgery by 5min. Sham operated mechanical VNS mice underwent cervical incisionfollowed by dissection of the underlying submandibular salivary glandsonly. The vagus nerve was neither exposed nor isolated.

Mice were injected with endotoxin (Escherichia coli LPS 0111:B4; Sigma)that was dissolved in sterile, pyrogen-free saline at stockconcentrations of 1 mg/ml. LPS solutions were sonicated for 30 minimmediately before use for each experiment. Mice received an LD₅₀ doseof LPS (7.5 mg/kg, i.p.). Blood was collected 2 h after LPSadministration, allowed to clot for 2 h at room temperature, and thencentrifuged for 15 min at 2,000×g. Serum samples were stored at −20° C.before analysis. TNF concentrations in mouse serum were measured byELISA (R & D Systems).

Mechanical VNS significantly reduced TNF production during lethalendotoxemia (FIG. 16). Compared with the control group, the mechanicalVNS group had a 75.8% suppression in TNF production (control=1819±181pg/ml vs. mechanical VNS=440±64 pg/ml, p=0.00003). These resultsindicate that mechanical nerve stimulation is sufficient to inhibitcytokine release.

EXAMPLE 6 Non-Invasive External Cervical Massage is Sufficient toActivate the Cholinergic Anti-Inflammatory Pathway

To determine whether mechanical VNS could be utilized in a non-invasive,transcutaneous manner to elicit anti-inflammatory effects, a model ofmurine cervical massage in lethal endotoxemia was developed. Mice wereanesthetized and positioned as described above. Following the midlinecervical incision, a unilateral left total submandibular sialoadenectomywas performed. No further dissection was performed, and the underlyingvagus nerve was not exposed. Following closure of the incision, animalsreceived external vagus nerve cervical massage using a cotton-tipapplicator. Cervical massage was performed using alternating directpressure applied antero-posteriorly adjacent to the left lateral borderof the trachea. Each pressure application was defined as onestimulation. The number of stimulations was quantified by frequency andtime. The lowest dose cervical massage group underwent 40 secstimulation at 0.5 stimulations s⁻¹. The middle dose cervical massagegroup underwent 2 min stimulation at 1 stimulations s⁻¹. The highestdose cervical massage group underwent 5 min stimulation at 2stimulations s⁻¹. Sham operated cervical massage mice underwentunilateral left submandibular sialoadenectomy only.

A dose response curve for TNF suppression was generated from thesestimulation groups and is shown in FIG. 17. The 40 sec (0.5 Hz) grouphad a 29.2% suppression of TNF (control=1879±298 pg/ml vs.massage=1331±503 pg/ml, p=0.38). The 2 min (1 Hz) group had a 36.8%suppression of TNF (control=1909±204 pg/ml vs. massage=1206±204 pg/ml,p=0.04). The 5 min (2 Hz) group had a 50.7% suppression of TNF(control=2749±394 pg/ml vs. massage=1355±152 pg/ml, p=0.02). These dataindicate that a non-invasive, easily performed, low risk, acceptedclinical therapeutic maneuver could be utilized to significantly reducesystemic inflammation.

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What is claimed is:
 1. A method for providing therapy to a patientsuffering from a condition mediated by an inflammatory cytokine cascade,wherein the method comprises: mechanically stimulating vagus nerveactivity by applying mechanical force comprising a plurality ofmechanical stimulations in an amount sufficient to inhibit theinflammatory cytokine cascade by at least 20% over the untreated state,and wherein the condition mediated by the inflammatory cytokine cascadeis selected from the group consisting of appendicitis, ulcerativecolitis, Crohn's disease, allergy, organ ischemia, reperfusion injury,sepsis, endotoxic shock, allograft rejection, arthritis, systemic lupuserythematosus, pancreatitis, peritonitis, hepatitis, burns, Behcet'ssyndrome, graft-versus-host disease, multiple sclerosis, atherosclerosisand cachexia; and monitoring a marker of the patient's inflammatorycytokine cascade.
 2. The method of claim 1, wherein the method comprisesmechanically stimulating efferent vagus nerve activity.
 3. The method ofclaim 1, wherein vagus nerve activity is stimulated using a vagus nervestimulator.
 4. The method of claim 1, wherein the marker comprises TNFlevel.
 5. A method for suppressing the production of TNF, wherein themethod comprises: identifying a patient suffering from a condition thatelevates TNF; monitoring TNF levels in the subject's blood; andmechanically stimulating the patient's vagus nerve by applyingmechanical force comprising a plurality of mechanical stimulations in anamount sufficient to decrease the amount of TNF in the subject's bloodby at least 20% over the untreated state.
 6. A method for providingtherapy to a patient suffering from a condition that causes aninflammatory cytokine cascade, wherein the method comprises: identifyinga patient suffering from a condition that causes inflammation throughthe inflammatory cytokine cascade; and evoking a sustained inhibition ofthe patient's inflammatory cytokine cascade by electrically stimulatingthe patient's vagus nerve for a first duration, followed by a secondduration during which the patient's vagus nerve is not electricallystimulated, wherein the second duration is at least three times longerthan the first duration, wherein at the end of the second duration thelevels of inflammatory cytokines are inhibited by at least 20% over theuntreated state.
 7. The method of claim 6, wherein the first duration isless than about 20 min.
 8. The method of claim 6, wherein the secondduration is greater than about 30 min.
 9. The method of claim 6, whereinthe patient's vagus nerve is stimulated in an amount sufficient toinhibit the inflammatory cytokine cascade without stimulating humoralanti-inflammatory mediators.
 10. The method of claim 9, wherein theelectrical stimulation of the patient's vagus nerve does not stimulate acorticosteroid or IL-10 response.
 11. The method of claim 6, furthercomprising electrically stimulating the patient's entire vagus nerve,including afferent and efferent nerves.
 12. The method of claim 6,further comprising electrically stimulating the patient's efferent vagusnerve by stimulating close to the target organ served by the efferentfibers or by directly stimulating the target organ.
 13. A method forproviding therapy to a patient suffering from a condition that causes aninflammatory cytokine cascade, wherein the method comprises: identifyinga patient suffering from a condition that causes inflammation throughthe inflammatory cytokine cascade; and mechanically stimulating thepatient's vagus nerve by applying mechanical force comprising aplurality of mechanical stimulations in an amount sufficient to inhibitthe inflammatory cytokine cascade by at least 20% over the untreatedstate.
 14. The method of claim 13, wherein the patient's vagus nerve ismechanically stimulated by pressure.
 15. The method of claim 13, whereinthe patient's vagus nerve is mechanically stimulated by vibration.
 16. Amethod of inhibiting an inflammatory cytokine cascade of a patientsuffering from a condition that causes an inflammatory cytokine cascade,wherein the method comprises: identifying a patient suffering from acondition that causes inflammation through the inflammatory cytokinecascade; and evoking a sustained inhibition of the patient'sinflammatory cytokine cascade by electrically stimulating the patient'svagus nerve for a first duration, followed by a second duration duringwhich the patient's vagus nerve is not electrically stimulated, whereinthe second duration is at least three times longer than the firstduration, wherein at the end of the second duration the levels ofinflammatory cytokines are inhibited by at least 20% over the untreatedstate.
 17. A method of inhibiting an inflammatory cytokine cascade of apatient suffering from a condition that triggers an inflammatorycytokine cascade, wherein the method comprises: identifying a patientsuffering from a condition that causes inflammation through theinflammatory cytokine cascade; and mechanically stimulating thepatient's vagus nerve by applying mechanical force comprising aplurality of mechanical stimulations in an amount sufficient to inhibitthe inflammatory cytokine cascade by at least 20% over the untreatedstate.
 18. A method of providing therapy to a patient suffering from acondition that causes an inflammatory cytokine cascade, wherein themethod comprises: identifying a patient suffering from a condition thatcauses inflammation through the inflammatory cytokine cascade;stimulating vagus nerve activity in an amount sufficient to inducesufficient secretion of a cholinergic agonist to inhibit theinflammatory cytokine cascade by at least 20% over the untreated state;and monitoring a marker of the patient's inflammatory cytokine cascade.19. The method of claim 18, the stimulating step further comprisingstimulating vagus nerve activity in an amount sufficient to inducesufficient secretion of acetylcholine.